Hoechst 33258 is a nuclear staining reagent that can penetrate cell membranes and stain DNA. It releases strong blue fluorescence after intercalating double-stranded DNA. Hoechst 33258 is often used for cell apoptosis detection. After staining, observe with a fluorescence microscope or detect with a flow cytometer. The excitation and emission wavelengths of Hoechst 33258-DNA are 352 nm and 461 nm, respectively. Instructions 1. For fixed cells or tissues 1) For cell or tissue samples, after fixation, wash them appropriately to remove fixative. Then, if immunofluorescence staining is required, perform immunofluorescence staining first, and then follow the subsequent steps for Hoechst 33258 staining after the staining is completed. If no other staining is required, the subsequent Hoechst 33258 staining is directly performed. 2) For adherent cells or tissue sections, add a small amount of Hoechst 33258 staining solution to cover the sample. For suspended cells, add at least 3 times the volume of the sample to be stained with staining solution and mix well. Leave at room temperature for 3-5 minutes. 3) Aspirate the Hoechst 33258 staining solution, wash 2-3 times with TBST, PBS or saline, 3-5 minutes each time. 4) Observe directly under a fluorescence microscope or after mounting the slide. When observing cell apoptosis, the nucleus of apoptotic cells will be densely stained, or fragmented and densely stained. 2. For living cells or tissues 5) Add an appropriate amount of Hoechst 33258 staining solution, which must fully cover the sample to be stained. Usually, 1ml staining solution is needed for one well of a six-well plate, and 100 microliters of staining solution is required for one well of a 96-well plate. 6) Incubate for 20-30 minutes at a temperature suitable for cell culture. Discard the staining solution, wash 2-3 times with PBS or culture solution to perform fluorescence detection. Storage conditions: Store at -20°C and protected from light, valid for one year. Precautions 1) Hoechst 33258 is irritating to the human body, please pay attention to proper protection. 2) Fluorescent dyes have the problem of quenching. It is recommended to complete the test on the same day after dyeing. 3) To slow down fluorescence quenching, anti-fluorescence quenching mounting solution can be used. 4) For your safety and health, please wear lab coats and disposable gloves for operation. | 
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